Accurate transmission of the genetic information requires complete duplication of the chromosomal DNA each cell division cycle. However, the idea that replication forks would form at origins of DNA replication and proceed without impairment to copy the chromosomes in the cell is too simple. The orderly progression of replication forks is challenged by encounters with template damage, slow moving and arrested RNA polymerases, and frozen DNA-protein complexes that stall the fork. Stalled forks are foci for genomic instability that causes genetic alterations and can give rise to cancer. Stalled forks must be remodeled/repaired and replication restarted/continued in order to maintain genomic stability. We have developed an Escherichia coli DNA replication system that allows us to analyze the consequences of collision of the replisome with leading-strand template damage. Using this system we have discovered that: (i) the E. coli replisome is inherently DNA damage- tolerant, capable of skipping over the leading-strand template lesion and restarting replication downstream; (ii) not all trans-lesion synthesis (TLS) DNA polymerases can interact successfully with the replisome to accomplish lesion bypass; (iii) replisome-mediated TLS competes with lesion skipping, and (iv) access to SFs of recombination proteins such as RecA, RecG, and RuvAB is different and that nascent strand regression catalyzed by these proteins at SFs is post-replicative, occurring once the replisome has moved downstream of the stall point. Using this replication system we can model all aspects of replisome stalling in vitro: the stall itself, replication restart by lesion skipping, polymerase uncoupling, lesion bypass, and remodeling of the SF by nascent strand regression. In this proposal we investigate the integrated network of responses to DNA damage that the bacterium uses to preserve genomic integrity. We ask: (i) how do stalled forks contribute to induction of the DNA damage (SOS) response? (ii) What is the mechanism of the UmuDC DNA replication checkpoint elaborated by the SOS response? (iii) What are the dynamics of exchange between DNA polymerase IV and DNA polymerase III during replisome-mediated trans-lesion bypass? (iv) How does DNA polymerase V interact with the replisome to accomplish trans-lesion bypass? And (v), how do replisomes overcome collisions with RNA polymerases that are themselves stalled by DNA template damage.